Functional and molecular identification of TREK-1 channel in myometrium in relati

描述（由申请人提供）：在大多数物种中，包括人类，17 - β -雌二醇和黄体酮的血浆水平在怀孕期间增加。妊娠后期女性类固醇激素的升高与子宫肌平滑肌的电重构有关。这些观察结果表明，雌性类固醇激素可以影响参与子宫平滑肌兴奋性的离子电导的活性和/或表达。虽然有报道称分娩机制与女性激素水平下降有关，但从离子通道表达角度研究产程收缩的机制尚不明确。在怀孕期间，子宫肌层的质量急剧增加，而宫内压力只有微小的增加。为了达到这个目的，子宫肌层的平滑肌在怀孕期间必须保持放松。因此，质膜拉伸激活的K+电导可能是子宫肌生成反应的重要组成部分。几个K+通道参与肌层静息膜电位和动作电位复极化的调节。最近有报道称，独特的K+通道由四个跨膜段和两个孔域（K2P）组成。在K2P通道中，TREK-1、TREK-2和TRAAK具有独特的功能特性，是最早克隆的拉伸激活K+通道。先前的研究表明，雌性类固醇激素影响了一些K+通道的转录表达。我们将在本提案中解决以下具体目标：目标1，我们将使用膜片钳方法确定小鼠肌层中SDK通道的功能表达，并将表征一氧化氮及其细胞内信号传导机制对这些通道的调节。目的2，我们将利用传统的微电极记录、等距力测量和Ca2+成像分析来表征完整肌层中拉伸依赖的超极化和弛缓。目的3，我们将使用分子和蛋白质技术研究TREK-1在小鼠肌层中的表达与天然SDK通道的关系。目的4，我们将通过表征TREK-1在未怀孕、怀孕、产后和去卵巢小鼠模型中的表达变化，了解不同雌性激素水平下天然SDK通道的功能表达。特别是本研究将扩大雌激素调节TREK-1通道导致不同程度的子宫内膜顺应性的分子机制。了解这一机制可能允许临床干预调节劳动和分娩，减少早产和随后的疾病的数量。公共卫生相关性：在过去20年中，早产率增长了30%以上。在所有早产病例中，大约40%的早产原因是未知的。本实验旨在评估子宫TREK-1通道随女性类固醇激素水平的急性和慢性变化而发生的变化，以研究其与妊娠和分娩机制的潜在功能关系。

DESCRIPTION (provided by applicant): In most species, including humans, plasma levels of 17beta-estradiol and progesterone increase during pregnancy. The elevation of female steroid hormones that accompanies the late stages of pregnancy has been linked to electrical remodeling of myometrial smooth muscle. These observations suggest that female steroid hormones can influence the activity and/or expression of ionic conductances involved in uterine smooth muscle excitability. Although it has been reported that the delivery mechanism is related to a decrease in female hormone levels, the mechanism of labor contraction has not been clearly studied in terms of ion channel expression. During pregnancy, the mass of myometrium increases dramatically, with only minimal increases in intrauterine pressure. To accomplish this, the smooth muscle of the myometrium must remain relaxed during pregnancy. Therefore, K+ conductances activated by stretch of the plasma membrane may contribute an important component of the myogenic response in the uterus. Several K+ channels participate in the regulation of resting membrane potential and repolarization of action potentials in the myometrium. Recently unique K+ channels have been reported that consist of four transmembrane segments and two-pore domains (K2P). Among K2P channels, TREK-1, TREK-2 and TRAAK have unique functional properties and represent the first cloned stretch-activated K+ channels. Previous studies suggest that female steroid hormones influence the transcriptional expression of a number of K+ channels. We will address the following specific aims in this proposal: Aim 1, we will identify SDK channel functional expression in the murine myometrium using patch-clamp methods and will characterize the regulation of these channels by nitric oxide and its intracellular signaling mechanisms. Aim 2, we will characterize stretch-dependent hyperpolarization and relaxation in intact myometrium using conventional microelectrode recordings, isometric force measurements and Ca2+ imaging analysis. Aim 3, we will use molecular and protein techniques to investigate TREK-1 expression in murine myometrium in relation to native SDK channels. Aim 4, we will characterize changes in TREK-1 expression in non-pregnant, pregnant, postpartum and ovariectomized murine models to understand the functional expression of native SDK channels under various levels of female hormones. In particular this investigation will expand the molecular mechanism of estrogen regulation of TREK-1 channels that leads to the different levels of myometrial compliance. Understanding this mechanism may allow for clinical intervention in the modulation of labor and delivery, reducing the number of premature births and subsequent disorders. PUBLIC HEALTH RELEVANCE: The rate of premature birth has grown by more than 30 percent in the last 20 years. In about 40 percent of all cases of preterm birth, the causes of preterm labor are unknown. Experiments outlined in this proposal are designed to evaluate changes in uterine TREK-1 channels that accompany acute and chronic changes in female steroid hormone levels to study a potential functional relationship with pregnancy and delivery mechanisms.